Structural mechanism for recognition of host receptor by botulinum neurotoxin An Abstract Botulinum neurotoxins (BoNTs), produced by the bacterium Clostridium botulinum, are the causative agents of neuroparalytic disease botulism. The extraordinary toxicity of BoNTs relies on the highly specific uptake of BoNTs by neuronal cells. A well-accepted dual-receptor model suggests that the cell surface binding and uptake process of BoNTs is mediated synergistically by specific protein receptors and gangliosides. BoNTs then exploit the synaptic vesicle recycling pathway entering the nerve terminus mediated by the protein receptors. However, it is largely unknown how various BoNTs develop serotype-specific mechanisms for protein receptor recognition, which is believed to account for the differences in BoNTs' biological activity. Our study is focused on BoNT/A serotype because it is a major concern for bioterrorism and is also the most commonly used medicine among the seven BoNT serotypes (BoNT/A-G). The goal is to understand the molecular mechanism by which BoNT/A specifically targets motoneurons through synaptic vesicle glycoprotein 2 that has three isoforms (SV2A, 2B, and 2C). Our preliminary studies show that BoNT/A and SV2C bind to each other through a relatively small protein-protein interface mostly involving backbone-backbone interactions, which is not sufficient to provide the high receptor binding affinity and specificity that BoNT/A needs. Remarkably, we found that BoNT/A takes advantage of SV2 glycosylation, a major form of post- translational modification of synaptic membrane proteins, to compensate for the shortfall on protein-mediated recognition. BoNT/A directly binds to an N-linked glycan of SV2, which is conserved in SV2A, 2B, and 2C and highly conserved across different vertebrates, to significantly enhance receptor binding affinity and specificity. This represents a new paradigm of intricate host-pathogen interactions. The specific aims are (1) to understand the structural basis for recognition of SV2C glycans by BoNT/A; and (2) to understand the affinity and specificity requirements for the BoNT/A-SV2 recognition. We will use an integrated approach that combines X-ray crystallography, site-directed mutagenesis, and binding assays. The achievement of our goal will guide the design of novel therapeutic approaches to prevent and treat botulism by preventing cell entry of BoNT/A, provide new insights into activity and side- effects of BoNT/A-based drugs, help improve their clinical efficacy, and suggest novel applications. Furthermore, this study will have impact on our basic biological understanding of the everlasting host-pathogen arms race, which may stimulate new ideas for therapeutic development.